1. Field of the Invention
The present application concerns the field of detection of ligand binding and more specifically pertains to high-speed instruments for automatically screening large numbers of potentially therapeutic compounds by measuring their relative binding affinity for various biological receptors.
2. Description of Related Art
The methods of genetic engineering have been successful in identifying the genetic sequence of disease-related cell surface receptors and artificially expressing these receptors in substantial quantity and in purified form. Such receptors can also be isolated from cell preparations. Combinatorial chemistry techniques provide rapid synthesis of low molecular weight compounds that are potential binding partners for isolated or identified receptors. Once created, these vast libraries of compounds must be screened for relative binding affinity to the target receptor. High speed machinery has become essential for type of testing or screening to be accomplished in a timely fashion.
The traditional approach to screening has been to label each compound, for example, with a radioactive moiety, a fluorescent tag, or a luminescent tag and to then determine binding of the labeled compound to the receptor of interest. For this purpose receptor is generally immobilized and the labeled compound is allowed to contact the receptor. After a period of time, unbound compound is washed away and the immobilized receptors are analyzed for the presence of the bound label.
There are at least two problems in this approach. First, washing tends to disrupt weak binding that may be important for detecting compounds with some but not ideal activity. Second, the task of labeling an entire library of perhaps hundreds of thousands of compounds presents a practical limit to the number of compounds that can be screened. Each compound presents a unique problem to be solved for labeling, and not all can be labeled by the same tag. Finally, the presence of the label can modify the affinity between the compound and the receptor.
Therefore, it would be highly desirable to have an instrument and a method that eliminates labeling and yet allows the quantitative analysis of the binding affinity between a compound and a receptor. This would remove a fundamental limit to high speed screening of very large libraries of compounds.
The present invention achieves this goal by providing an instrument and a method that detects a change in the optical characteristics of the solid support to which the receptor has been bound when the receptor is occupied by an unlabeled binding compound. The solid support is a specific type of colloidal particle of less than 100 nm in diameter. This small size means that the binding kinetics for the reaction are similar to solution phase kinetics. The instrument automatically mixes the compound and the colloidal suspension of receptors, incubates the mixture, then reads the result at the rate of thousands of tests per hour.
In the copending application referenced above the present inventors discovered that optical resonance could be used to detect the crosslinking of particles. The current invention is not dependent on crosslinking of particles. As developed below, the present invention directly detects the binding of an unlabeled ligand to a particle.
The invention utilizes a specialized type of optically sensitive, sub-micron particle, the surface of which is pre-coated with a monolayer of a specific molecular receptor that can bind solution phase ligands (In this invention, xe2x80x9creceptorxe2x80x9d refers to a molecule immobilized on the particle that is capable of binding other molecules that are in free solution. Furthermore, in this invention, the molecules in free solution are termed xe2x80x9cligandsxe2x80x9d). The particle itself is a transducer that directly senses the binding of ligands to the receptor and creates an optical signal indicative of this binding. The particle itself is the binding sensor, thereby obviating the need to label the ligand.
The signal transduction properties of the particles used in the invention depend upon there being an optical light scatter and absorption resonance at a specific resonance wavelength xcexR. Such a resonance is present in small particles having a complex refractive index wherein a real part n(xcex) of the index approaches 0 while an imaginary part k(xcex) approaches 2 simultaneously at the specific wavelength xcexR. (In this invention a small particle is one that is less than approximately one tenth the wavelength of the incident light.) It is known from detailed theories of light scatter, that, when the above resonance condition is met, both light scatter and absorption are substantially greater than predicted by the Rayleigh theory of simple light scatter where it is assumed that n and k are constant and not functions of xcex (Bohrens and Huffman). The present invention demonstrates the additional surprising result that the intensity of small particle light scatter and absorption at the resonance wavelength changes when ligands bind to receptors immobilized on such resonant particles. The closer the two conditions of n and k are met, the stronger is the resonance, and the more sensitive the receptor-coated particles are to ligand binding.
Normally, expressions for light scatter and absorption from small particles with constant refractive indices are simple functions of wavelength. Generally speaking, both light scatter and absorption are highest for short wavelengths (e.g. for ultra-violet light) and lowest for long wavelengths (e.g. red or infrared light). This wavelength dependence is monotonic, meaning that there is a continuous progression from intense absorption and scatter at short wavelengths to less intense absorption and scatter at long wavelengths. Exceptions to this behavior occur for particles with a complex refractive index that has strong wavelength dependence.
If the refractive index of the particle varies with wavelength in such a way that the two conditions; 1). n(xcex) approaches 0 and 2). k(xcex) approaches 2 are simultaneously met at a specific wavelength xcexR, then light scatter and absorption increase dramatically in a narrow wavelength band around xcexR. This departure is termed a resonance. The resonance is delicate, and can be perturbed by the deposition of chemical agents on the surface of the particle. When a layer of receptors is coated onto the particle, the resonance is altered; but it is a surprising and important aspect of the present invention that when ligands then bind to the receptor layer, there is a further perturbation of the resonance that can be readily detected optically.
The optical signal pertaining to this invention is detected either by absorption or light scatter photometry. The optical resonance increases the level of light scatter and absorption by at least a factor of ten above the normal levels of light scatter at the resonance wavelength xcexR. This enables the optical detection of particles that are as small as the order of 100 xc3x85ngstroms (10 nm) at low concentrations. Such small particles advantageously diffuse virtually as rapidly as macromolecules in solution. Such rapid particle motion coupled with an interparticle spacing that is of the order of micrometers (xcexcm) means that the ligand receptor surface binding reaction occurs at nearly maximal rates. Such particle-based reactions should be contrasted to binding reactions that occur on flat surfaces such as when receptors are immobilized on the surface of micro-wells. There the ligand must diffuse over distances of about a millimeter (1000 xcexcm) or more to reach the receptor. These latter reactions are slow and impede the desired high throughput of an automated system for binding reaction screening.
The binding reactions and signal transduction in the present invention occur in a single, rapid step. This enables binding assays to be easily automated and carried out at such system throughput rates that large collections of ligands and receptors can be evaluated for binding affinity in relatively short periods of time by machines that run continuously and benefit from low complexity fluid handling mechanisms.